Structure and Localization of the Flavonoid Multienzyme Complex

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Interactions between biological macromolecules underlie virtually every cellular process, including the organization of cooperating enzymes into macromolecular complexes. Multienzyme systems provide the means to attain high local substrate concentrations, regulate competition among branch pathways, coordinate the activities of interdependent pathways, and sequester toxic or volatile intermediates within the cell. Yet remarkably little is known about the architecture of any enzyme complex or the molecular mechanisms that direct the interaction of the individual components. The project will build on previous work in the Winkel laboratory to characterize the structure and function of the flavonoid enzyme complex in Arabidopsis thaliana, including the use of small angle neutron scattering to develop a three-dimensional model of this system. Several new technologies for studying interaction interfaces will be tested in an effort to confirm proposed interactions in planta, and to identify factors that may arbitrate assembly of the complex. The determinants of the recently discovered differential localization of flavonoid enzymes will also be characterized. These studies will contribute to a general understanding of how macromolecular interactions occur with the requisite specificity and flexibility to effectively mediate biological processes in the crowded and dynamic cell interior. This knowledge is essential for the effective application of metabolic engineering to the agronomic and nutritional improvement of plants and for the bioproduction of valuable metabolites in microorganisms, plants, and animals.The project will provide a training ground in contemporary molecular genetics, biochemistry, and computational biology for undergraduates, graduate students, and a postdoctoral fellow. The broader impact of this work also involves a specific effort to study the outcomes and impacts of research experiences for undergraduates on the participating students and their mentors. Some 35 undergraduate students have conducted research in the Winkel laboratory over more than a decade; this provides an ideal scenario for studying these experiences in a rigorous and broadly applicable way. An assessment will be made of the academic and career opportunities that undergraduates pursue following their research experience, the extent to which the research experience prepare and/or encourage undergraduates to pursue academic and career opportunities, and the extent to which research experiences for undergraduates impacted the mentors.